Recombinant protein capable of binding specifically and quickly to troponin i derived from human myocardium

ABSTRACT

Provided is a recombination protein which binds specifically to troponin I derived from human myocardium. The recombinant protein includes a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 63; and a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 65.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of International Application No.PCT/JP2012/001726, with an international filing date of Mar. 13, 2012,which claims priority of Japanese Patent Application No. 2011-275413,filed on Dec. 16, 2011, the entire contents of each of which areincorporated herein by reference.

FIELD

The technical field relates to a recombinant protein capable of bindingspecifically and quickly to troponin I derived from human myocardium.

BACKGROUND

Non Patent Literature 1 and Non Patent Literature 2 disclose that aconcentration of troponin I derived from myocardial tissue increasesrapidly in the blood of a patient who has suffered acute myocardialinfarction.

CITATION LIST Non Patent Literature

-   Non Patent Literature 1-   Aleksei G. Katrukha et. al., “Troponin I is released in bloodstream    of patients with acute myocardial infarction not in free form but as    complex”, Clinical Chemistry, Vol. 43, Issue 8, p.p. 1379-1385    (1997)-   Non Patent Literature 2-   Till Keller et. al., “Sensitive Troponin I Assay in Early Diagnosis    of Acute Myocardinal Infarction”, The NEW ENGLAND JOURNAL of    MEDICINE, Vol. 361, pages 868-877 (2009)

SUMMARY

One non-limiting and exemplary embodiment provides a recombinant proteincapable of binding specifically and quickly to troponin I derived fromhuman myocardium.

Additional benefits and advantages of the disclosed embodiments will beapparent from the specification and Figures. The benefits and/oradvantages may be individually provided by the various embodiments andfeatures of the specification and drawings disclosure, and need not allbe provided in order to obtain one or more of the same.

In one general aspect, the techniques disclosed here feature arecombinant protein which binds specifically to troponin I derived fromhuman myocardium. The recombinant protein includes a light chainvariable region consisting of the amino acid sequence represented by SEQID NO: 63, and a heavy chain variable region consisting of the aminoacid sequence represented by SEQ ID NO: 65.

The present disclosure provides a recombinant protein capable of bindingspecifically and quickly to troponin I derived from human myocardium.

The recombinant protein and the method of the present disclosure can beused for early detection of acute myocardial infarction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an antibody.

FIG. 2 shows a PCR method in the step (c-2).

DETAILED DESCRIPTION

The embodiment of the present disclosure is described below.

Explanation of Terms

First, the terms used in the present specification are described.

FIG. 1 shows an antibody. The antibody 1 has a letter “Y” shape. Theantibody 1 has two Fab regions and one Fc region. The antibody 1consists of two heavy chains 2 and two light chains 3. Each heavy chain2 consists of a heavy chain constant region 1 (22), a heavy chainconstant region 2 (23), a heavy chain constant region 3 (24) and a heavychain variable region 21. Each light chain 3 consists of a light chainvariable region 31 and a light chain constant region 32.

Each Fab region consists of the one heavy chain variable region 21, theone heavy chain constant region 1 (22), the one light chain variableregion 31, and the one light chain constant region 32. The light chain 3is connected to the heavy chain 2 through a linker 4. The one heavyvariable region 21 is present at the end of the heavy chain 2. The onelight chain variable region 31 is present at the end of the light chain3. An antigen is specifically bound to the antibody 1. In more detail,the antigen is bound specifically to the Fv region, which consists ofthe heavy chain variable region 21 and the light chain variable region31. In the present specification, the antigen is troponin I derived fromhuman myocardium.

The recombination protein according to the embodiment includes the lightchain variable region 31 consisting of the amino acid sequencerepresented by SEQ ID NO: 63 and the heavy chain variable region 21consists of the amino acid sequence represented by SEQ ID NO: 65. Therecombinant protein of the present disclosure binds specifically andquickly to the troponin I derived from human myocardium.

The recombinant protein of the present disclosure may be either anantibody or an antibody fragment.

The antibody has a shape of the form of the letter “Y” shown in FIG. 1.The light chain variable region 31 and the heavy chain variable region21, both of which is included in the antibody of the present disclosure,consist of amino acid sequences represented by SEQ ID NO: 63 and SEQ IDNO: 65, respectively. In the antibody, the light chain variable region31 is connected to the heavy chain variable region 21 through a linker(not shown).

Examples of the antibody fragment include a Fab antibody fragment, aF(ab′)₂ antibody fragment and an scFv antibody fragment.

The Fab antibody fragment consists of one Fab region. In other words,the Fab antibody fragment consists of the one light chain variableregion 31 (SEQ ID NO: 63), the one heavy chain variable region 21 (SEQID NO: 65), the one light chain constant region 32, the one heavy chainconstant region 1 (22), and the linker 4. The light chain constantregion 32 is connected to the heavy chain constant region 1 (22) throughthe linker 4.

The F(ab′)₂ antibody fragment consists of two Fab regions. As above,each Fab region consists of the one light chain variable region 31 (SEQID NO: 63), the one heavy chain variable region 21 (SEQ ID NO: 65), theone light chain constant region 32, the one heavy chain constant region1 (22), and the linker 4. These two Fab regions are connected to eachother through another linker (not shown). For example, one heavy chainconstant region 1 (22) is connected to the other heavy chain constantregion 1 (22) through another linker (not shown).

The scFv antibody fragment consists of the light chain variable region31 (SEQ ID NO: 63), the heavy chain variable region 21 (SEQ ID NO: 65),and a linker. The light chain variable region 31 (SEQ ID NO: 63) isconnected to the heavy chain variable region 21 (SEQ ID NO: 65) througha linker (not shown).

As long as the recombination protein is capable of binding specificallyand quickly to troponin I derived from human myocardium, the linkerconnecting the light chain variable region 31 (SEQ ID NO: 63) and theheavy chain variable region 21 (SEQ ID NO: 65) is not specificallylimited. An example of the linker is a peptide consisting of 5-20 aminoacids. For example, the linker is a peptide consisting of the amino acidsequence represented by GGGGSGGGGSGGGGS (SEQ ID NO: 64). Another exampleof the linker is a disulfide bond (-sulfur atom (S)—sulfur atom (S)—).

As long as the recombination protein is capable of binding specificallyand quickly to troponin I derived from human myocardium, the N-terminalof the light chain variable region 31 (SEQ ID NO: 63) may be modifiedwith an amino acid sequence. The C-terminal thereof may be alsomodified.

As long as the recombination protein is capable of binding specificallyand quickly to troponin I derived from human myocardium, the N-terminalof the heavy chain variable region 21 (SEQ ID NO: 65) may be modifiedwith an amino acid sequence. The C-terminal thereof may be alsomodified. An example of the amino acid sequence to modify the C-terminalof the heavy chain variable region 21 (SEQ ID NO: 65) is AAALEHHHHHH(SEQ ID NO: 66).

When the recombinant protein of the present disclosure is brought intocontact with troponin I derived from human myocardium, the recombinantprotein of the present disclosure binds specifically and quickly to thetroponin I derived from human myocardium. For example, when therecombinant protein of the present disclosure is mixed with troponin Iderived from human myocardium, the recombinant protein of the presentdisclosure binds specifically and quickly to the troponin I derived fromhuman myocardium.

Detection of the binding of the recombinant protein of the presentdisclosure to the troponin I derived from human myocardium can becarried out by methods for detecting antigen-antibody binding which arewell known to those skilled in the art. Examples of such methods includethe ELISA sandwich method.

The recombinant protein of the present disclosure can be produced usingan ordinal protein expression technique. For example, first, a vectorincluding a gene sequence coding for the recombinant protein of thepresent disclosure is prepared. An example of the vector is a plasmid.Then, cells (e.g., Escherichia coli) are transformed with this vector.These cells are incubated to produce the recombinant protein of thepresent disclosure.

In order to obtain the scFv antibody fragment efficiently, it isbeneficial that the recombinant protein of the present disclosure isproduced by a refolding method. Non Patent Literature 3 discloses therefolding method.

-   Non Patent Literature 3-   Jun Kamishikiryo et. al., “Molecular Basis for LLT1 Protein    Recognition by Human CD161 Protein (NKRP1A/KLRB1)”, THE JOURNAL OF    BIOLOGICAL CHEMISTRY, VOL. 286, NO. 27, p.p. 23823-23830.

Examples of the technique of the present disclosure are as follows.

1st aspect: A recombinant protein which binds specifically to troponin Iderived from human myocardium. The recombinant protein includes a lightchain variable region comprising the amino acid sequence represented bySEQ ID NO: 63, and a heavy chain variable region comprising the aminoacid sequence represented by SEQ ID NO: 65.

2nd aspect: In the recombinant protein according to the 1st aspect, therecombinant protein may be an antibody.

3rd aspect: In the recombinant protein according to the 1st aspect, therecombinant protein may be an antibody fragment.

4th aspect: In the recombinant protein according to the 3rd aspect, theantibody fragment may be a Fab antibody fragment.

5th aspect: In the recombinant protein according to the 3rd aspect, theantibody fragment may be a F(ab′)₂ antibody fragment.

6th aspect: In the recombinant protein according to the 3rd aspect, theantibody fragment may be an scFv antibody fragment.

7th aspect: A method for binding a recombinant protein specifically totroponin I derived from human myocardium, includes the following steps.A step (a) is a step of preparing the recombinant protein. Therecombinant protein includes a light chain variable region consisting ofthe amino acid sequence represented by SEQ ID NO: 63, and a heavy chainvariable region consisting of the amino acid sequence represented by SEQID NO: 65. A step (b) is a step of bringing the recombinant protein intocontact with the troponin I derived from human myocardium to bind therecombinant protein specifically to the troponin I derived from humanmyocardium. In this aspect, for example, the step (b) may be carried outin vitro. 8th aspect: In the method according to the 7th aspect, therecombinant protein may be an antibody.

9th aspect: In the method according to the 7th aspect, the recombinantprotein may be an antibody fragment.

10th aspect: In the method according to the 9th aspect, the antibodyfragment may be a Fab antibody fragment.

11th aspect: In the method according to the 9th aspect, the antibodyfragment may be a F(ab′)₂ antibody fragment.

12th aspect: In the method according to the 9th aspect, the antibodyfragment may be scFv antibody fragment.

EXAMPLES

An example for supporting an exemplary embodiment of the presentdisclosure is described below.

Example 1

Table 1, Table 2, Table 3, and Table 4 show the primers used in Example1.

Table 1 shows the forward mixture primers (primers 1-21, SEQ ID NOS:02-22) for amplifying a light chain variable region.

Table 2 shows the forward mixture primers (primers 22-44, SEQ ID NOS:23-45) for amplifying a heavy chain variable region.

Table 3 shows the reverse mixture primers (primers 45-49, SEQ ID NOS:46-50) for amplifying a light chain variable region.

Table 4 shows the reverse mixture primers (primers 50-55, SEQ ID NOS:51-56) for amplifying a heavy chain variable region.

TABLE 1 Primer 1 SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG 02TWCTCWCCCARTC Primer 2 SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTS03 TGMTSACYCAGTC Primer 3 SEQ ID NO:CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG 04 TGMTMACTCAGTC Primer 4SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG 05 TGHTRWCACAGTCPrimer 5 SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG 06TRATGACMCAGTC Primer 6 SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTM07 AGATRAMCCAGTC Primer 7 SEQ ID NO:CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTC 08 AGATGAYDCAGTC Primer 8SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTT 09 TGCTGACTCAGTCPrimer 9 SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG 10TTCTCAWCCAGTC Primer 10 SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG11 WGCTSACCCAATC Primer 11 SEQ ID NO:CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTS 12 TRATGACCCARTC Primer 12SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYRTTK 13 TGATGACCCAVACPrimer 13 SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATYC 14AGATGACACAGAC Primer 14 SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG15 TGATGACACAACC Primer 15 SEQ ID NO:CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATCC 16 AGCTGACTCAGCC Primer 16SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG 17 TGATGACBCAGKCPrimer 17 SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG 18TGATAACYCAGGA Primer 18 SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG19 TGATGACCCAGWT Primer 19 SEQ ID NO:CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYGTGS 20 TGMTSACYCAGTC Primer 20SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYGCTG 21 TTGTACTCAGGAATCPrimer 21 SEQ ID NO: CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG 22TDHTVWCHCAGTC

TABLE 2 Primer 22 SEQ ID NO: AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG 23AKGTRMAGCTTCAGGAGYC Primer 23 SEQ ID NO:AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG 24 AGGTNCAGCTBCAGCAGTC Primer 24SEQ ID NO: AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCC 25 AGGTGCAGCTGAAGSASTCPrimer 25 SEQ ID NO: AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCC 26AGSTBCAGCTGCAGCAGTC Primer 26 SEQ ID NO:AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG 27 AGGTYCAGCTYCAGCAGTC Primer 27SEQ ID NO: AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG 28 ARGTCCARCTGCAACARTCPrimer 28 SEQ ID NO: AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCC 29AGGTYCAGCTBCAGCARTC Primer 29 SEQ ID NO:AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCC 30 AGGTYCARCTKCAGCAGTC Primer 30SEQ ID NO: AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCC 31 AGGTCCACGTGAAGCAGTCPrimer 31 SEQ ID NO: AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG 32AGGTGAASSTGGTGGARTC Primer 32 SEQ ID NO:AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG 33 AVGTGAWGYTGGTGGAGTC Primer 33SEQ ID NO: AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG 34 AGGTGAAGGTCATCGAGTCPrimer 34 SEQ ID NO: AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCS 35AGGTGCAGSKGGTGGAGTC Primer 35 SEQ ID NO:AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG 36 AKGTGCAMCTGGTGGAGTC Primer 36SEQ ID NO: AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG 37 AAGTGCAVCTGGTGGAGTCPrimer 37 SEQ ID NO: AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG 38AGGTGAAGCTGATGGARTC Primer 38 SEQ ID NO:AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG 39 AGGTGCARCTTGTTGAGTC Primer 39SEQ ID NO: AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG 40 ARGTRAAGCTTCTCGAGTCPrimer 40 SEQ ID NO: AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG 41AAGTGAARSTTGAGGAGTC Primer 41 SEQ ID NO:AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG 42 AAGTGATGCTGGTGGAGTC Primer 42SEQ ID NO: AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCC 43 AGGTTACTCTRAAAGWGTSTGPrimer 43 SEQ ID NO: AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCC 44AGGTCCAAYTVCAGCARCC Primer 44 SEQ ID NO:AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG 45 ATGTGAACTTGGAAGTGTC

TABLE 3 Primer 45 SEQ ID NO: ACCAGAGCCGCCGCCGCCGCTACCACCACCACCC 46CGTTTGATTTCCARCTTKG Primer 46 SEQ ID NO:ACCAGAGCCGCCGCCGCCGCTACCACCACCACCC 47 CGTTTTATTTCCAGCTTGG Primer 47SEQ ID NO: ACCAGAGCCGCCGCCGCCGCTACCACCACCACCC 48 CGTTTSAGCTCCAGCTTGGPrimer 48 SEQ ID NO: ACCAGAGCCGCCGCCGCCGCTACCACCACCACCC 49CGTTYWATTTCCAACTTWG Primer 49 SEQ ID NO:ACCAGAGCCGCCGCCGCCGCTACCACCACCACCC 50 CCTAGGACAGTCAGTTTGG

TABLE 4 Primer 50 SEQ ID CGGCACCGGCGCACCTGCGGCCGCYGAGGAAACGGTGAC NO: 51CGTGGT Primer 51 SEQ ID CGGCACCGGCGCACCTGCGGCCGCYGAGGAGACTGTGAG NO: 52AGTGGT Primer 52 SEQ ID CGGCACCGGCGCACCTGCGGCCGCYGAGGAGACGGTGAC NO: 53TGAGRT Primer 53 SEQ ID CGGCACCGGCGCACCTGCGGCCGCYGAGGAAGACTGTAG NO: 54AGTGGT Primer 54 SEQ ID CGGCACCGGCGCACCTGCGGCCGCYGCGGAGACASTGAC NO: 55CAGAGT Primer 55 SEQ ID CGGCACCGGCGCACCTGCGGCCGCYGCAGAGACASTGAC NO: 56CAGAGT

Step (a-1) Preparation of a Hybridoma (Derived from Mouse Spleen)Capable of Producing Monoclonal Antibodies which Specifically Bind toTroponin I Derived from Human Myocardium

A peptide having an amino acid sequence (SEQ ID NO: 01, purchased fromSigma Aldrich Japan Co., Ltd.,RPAPAPIRRRSSNYRAYATEPHAKKKSKISASRKLQLKTLLLQIAK) contained in troponin Iderived from human myocardium was connected to human serum albumin(purchased from Sigma Aldrich Japan Co. Ltd.) using a sulfo-SMCC crosslinker (purchased from Servo Fischer Scientific Co., Ltd.).

More particularly, the sulfo-SMCC cross linker (0.5 mg) was dissolved in100 microliters of a phosphate buffered saline so as to obtain a firstaqueous solution. This first aqueous solution was left under atemperature of 50 degrees Celsius for ten minutes.

The human serum albumin (10 mg) was dissolved in one milliliter of aphosphate buffered saline to obtain a second aqueous solution.

The first aqueous solution was mixed with the second aqueous solution toobtain a mixture. The mixture was left at rest for 30 minutes. In thisway, the sulfo-SMCC cross linker was connected to the human serumalbumin.

The mixture was passed through a column (purchased from GE health care,trade name: PD10) to remove the unreacted sulfo-SMCC cross linker.

The above-mentioned peptide (SEQ ID NO: 01, 1.5 mg) was dissolved indimethylsulfoxide (hereinafter, referred to as “DMSO”) to obtain a DMSOsolution. The DMSO solution (100 microliters) was added to the mixture(1 mL) having a concentration of 2 mg/ml. Afterwards, the mixture wasleft overnight to connect the sulfo-SMCC cross linker to the peptide(SEQ ID NO: 01).

In this way, human serum albumin modified with the peptide having theamino acid sequence (SEQ ID NO: 01) contained in the troponin I wasobtained. Hereinafter, this human serum albumin is referred to as“troponin-modified HSA”.

A complete Freud adjuvant (purchased from Wako Pure Chemical IndustriesCo., Ltd.) and the troponin-modified HSA were mixed to obtain a mixture.This mixture was injected to a BALB/c mouse. The BALB/c mouse is a kindof albino mouse.

Two weeks later, a mixture of phosphate buffered saline (hereinafter,referred to as “PBS”) and troponin-modified HSA was injected into theBALB/c mouse. This was repeated once again. In this way, the BALB/cmouse was immunized by troponin-modified HSA for one month. In otherwords, by administering the mixture to the BALB/c mouse, antibodiesagainst troponin-modified HSA were produced in the body of the BALB/cmouse.

The spleen of the immunized BALB/c mouse was taken out. In accordancewith the cell fusion method disclosed in Non Patent Literature 4,hybridomas were obtained. Afterwards, the hybridomas were incubated in aculture fluid. The number of hybridomas (cells) after the incubation wasapproximately 5×10⁶. The hybridomas obtained in this way were capable ofproducing the monoclonal antibody which specifically bound to troponin Iderived from human myocardium.

-   Non Patent Literature 4-   G. Kohler et al., Nature, 256, 495 (1975)

Step (a-2) Extraction of Total Mouse RNAs from the Hybridoma Cells

In order to destroy the cell membrane of the cultured hybridomas, onemilliliter of TRIzol (Purchased from Invitrogen Co., Ltd.) was added tothe culture fluid containing the hybridomas, and the culture fluid wasstirred well.

Then, a chloroform liquid having a volume of 0.2 mL (degree of purity:99.9%) was added to the culture fluid, and the culture fluid was stirredwell again.

The culture fluid was subjected to a centrifugal separation at anacceleration of gravity of 117600 m/s² under a temperature of 4 degreesCelsius for 15 minutes. The supernatant (500 μL) was acquired.Isopropanol (500 μL) was added to the obtained supernatant and left atrest under room temperature for ten minutes.

The culture fluid was again subject to a centrifugal separation having acondition identical to the above-mentioned condition to obtain aprecipitate. A seventy-five percent ethanol aqueous solution (1 mL) wasadded to the obtained precipitate so as to obtain an ethanol solution.

The ethanol solution was subjected to a centrifugal separation at anacceleration of gravity of 73500 m/s² for five minutes. The precipitatewas dried. In this way, total mouse RNAs were obtained.

Step (b-1) Extraction of mRNA from the Total Mouse RNAs

Using an Oligotex™-dT30 <Super> mRNA Purification kit (purchased fromTakara bio Co., Ltd.), mRNA was extracted from the total mouse RNAsobtained in the step (a-2).

RNase-free water (100 μL) was injected into a microtube. This microtubewas set at a block incubator (purchased from ASTEC CO. LTD.) and heatedunder a temperature of 70 degrees Celsius for one hour.

The total mouse RNAs were dissolved in the RNase-free water (100 μL).

A 2× binding buffered solution (100 μL) included in the kit and anoligotex (10 μL) included in the kit were mixed with the RNase-freewater (100 μL). Subsequently, the mixture was left at rest under atemperature of 70 degrees Celsius for three minutes. Furthermore, themixture was left at rest under room temperature for ten minutes.

The mixture was subjected to a centrifugal separation at an accelerationof gravity of 147000 m/s² for five minutes. The supernatant was removed,and the precipitate was suspended in Wash buffer (350 μL) included inthe kit. The suspension liquid was supplied to a column included in thekit. The column was subjected to a centrifugal separation at anacceleration of gravity of 147000 m/s² for 30 seconds.

The Wash buffer (350 μL) was supplied to the column to wash the column.The column was subjected to a centrifugal separation at an accelerationof gravity of 147000 m/s² again for 30 seconds.

A microtube for sample collection was attached to the bottom of thecolumn.

In order to extract mRNA contained in the column, RNase-free water (20μL) contained in the microtube was supplied to the column. Subsequently,the column was subjected to a centrifugal separation at an accelerationof gravity of 147000 m/s² for three minutes. Again, RNase-free water (20μL) was supplied to the column, and the column was subjected to acentrifugal separation at an acceleration of gravity of 147000 m/s² forthree minutes.

Thus, the extract liquid containing the mRNA was obtained in themicrotube.

(Step b-2) Reverse-Transcription from mRNA to cDNA

The mRNA contained in the obtained extract liquid wasreverse-transcripted with a reverse-transcriptase (purchased from Takarabio Co., Ltd, trade name: Primersript) to obtain a solution containingcDNA.

Step (b-3-1) Amplification of the Gene Coding for the Light ChainVariable Region Using the cDNA

The gene fragment (SEQ ID NO: 58, hereinafter, referred to as “VL genefragment”) coding for the light chain variable region of theabove-mentioned monoclonal antibody was amplified by a PCR method usingthe cDNA contained in the solution, the forward primers 1-21 (SEQ IDNOS: 02-22), and the reverse primers 1-5 (SEQ ID NOS: 23-27). Thepolymerase used in this PCR method was purchased from Takara bio Co.,Ltd under a trade name of TaKaRa Ex Taq Hot start Version.

The protocol of this PCR method is shown in Table 5.

TABLE 5 One cycle ninety six degrees Celsius for thirty seconds fiftytwo degrees Celsius for one minute sixty eight degrees Celsius for oneminute

The number of the cycles: 35.

Finally, the solution was left at 68 degrees Celsius for four minutes.In this way, a PCR solution was obtained. This PCR solution containedthe amplified VL gene fragment (SEQ ID NO: 58).

For the confirmation and purification of the amplified VL gene fragment,the obtained PCR solution was subjected to an electrophoresis using agel containing agarose having a concentration of 2% by weight.

Step (b-3-2) Amplification of the Gene Coding for the Heavy ChainVariable Region Using the cDNA

The gene fragment (SEQ ID NO: 57, hereinafter, referred to as “VH genefragment”) coding for the heavy chain variable region of theabove-mentioned monoclonal antibody was amplified by a PCR method usingthe cDNA contained in the solution, the forward primers 22-44 (SEQ IDNOS: 28-50), and the reverse primers 6-11 (SEQ ID NOS: 51-56). Thepolymerase used in this PCR method was purchased from Takara bio Co.,Ltd under a trade name of TaKaRa Ex Tag Hot start Version.

The protocol of this PCR method was identical to that used for the VLgene fragment.

Finally, the solution was left at 68 degrees Celsius for four minutes.In this way, a PCR solution was obtained. This PCR solution containedthe amplified VH gene fragment (SEQ ID NO: 57).

For the confirmation of the generation of the VH gene fragment and forthe purification of the VH gene fragment, the obtained PCR solution wassubjected to an electrophoresis using a gel containing agarose having aconcentration of 2% by weight.

Step (b-4) Connection of the VL Gene Fragment and the VH Gene Fragment

The purified VH gene fragment (SEQ ID NO: 57) was connected to thepurified VL gene fragment (SEQ ID NO: 58) using an overlap extension PCRmethod. In this way, the gene fragment (SEQ ID NO: 59, hereinafter,referred to as “scFv gene fragment”) coding for the scFv antibodyfragment of the above-mentioned monoclonal antibody was obtained. Theobtained gene fragment (SEQ ID NO: 59) was modified with restrictionenzyme sites Nco1 and Not 1 at the 5′-end and 3′-end thereof,respectively.

Step (c-1) Introduction of the Gene to a Vector

The scFv gene fragment was ligated into a protein expression vector(purchased from Takara bio Co., Ltd, trade name: pET22b(+)). The detailof the ligation is described below.

First, the scFv gene fragment was treated with restriction enzymes Nco1and Not1 (both of which were purchased from Takara bio Co., Ltd.). ThescFv gene fragment was purified by an electrophoresis method to obtainan aqueous solution containing the scFv gene fragment.

The protein expression vector was also treated with restriction enzymesNco1 and Not1 (both of which were purchased from Takara bio Co., Ltd.).The protein expression vector was also purified by an electrophoresismethod to obtain an aqueous solution containing the protein expressionvector.

These two aqueous solutions were mixed to obtain a mixture.

An enzyme (purchased from Toyobo Co., Ltd., trade name: Ligation Highver. 2) was added to the mixture, and the mixture was left under atemperature of 16 degrees Celsius for two hours. In this way, the scFvgene fragment was ligated into the protein expression vector.

Escherichia coli cells (purchased from Takara bio Co., Ltd., trade name;DH5a competent cell) were transformed with the protein expression vectorin which the scFv gene fragment was thus ligated.

Subsequently, the Escherichia coli cells were incubated for sixteenhours on an LB plate culture medium containing ampicillin having aconcentration of 100 μg/mL. After the incubation, a single colony formedon the LB plate culture medium was picked up. The single colony wassupplied to an LB liquid culture medium (5 mL) containing ampicillinhaving a concentration of 100 μg/mL, and the colony was incubated for 16hours.

In order to remove an unnecessary gene sequence included in the proteinexpression vector pET22b(+), the protein expression vector pET22b(+) wasextracted from this LB liquid culture medium using a kit (QIAGEN Co.,Ltd. trade name: QIAprep spin miniprep kit). By a PCR method using theextracted protein expression vector pET22b(+), the primer 56 (SEQ ID NO:67), and the primer 57 (SEQ ID NO: 68), the signal sequence (DNAsequence, SEQ ID NO: 60) of the protein expression vector pET22b(+) wasremoved. Thus, the expression vector coding for the wild type scFvantibody fragment was obtained.

Step (c-2) Introduction of the Mutations to the Vector

The expression vector obtained in the step (c-1) included the scFv genefragment (SEQ ID NO: 59). Among the 729 bases constituting the scFv genefragment (SEQ ID NO: 59) included in the expression vector, seven baseswere substituted. In more detail, the 589th adenine (A), the 590thcytosine (C), the 607th thymine (T), the 608th cytosine (C), the 609thcytosine (C), the 613th adenine (A), and the 615th cytosine (C) weresubstituted with guanine (G), adenine (A), guanine (G), adenine (A),thymine (T), guanine (G), and thymine (T), respectively.

More particularly, as shown in FIG. 2, a PCR method using the primer 58(SEQ ID NO: 69), the primer 59 (SEQ ID NO: 70) and the expression vectorobtained in the step (c-1) was performed. The primer 58 (SEQ ID NO: 69)was complementary to the gene sequence from 579^(th) base to 626^(th)base included in the scFv gene fragment (SEQ ID NO: 59) except for theseven bases to be substituted. The primer 59 (SEQ ID NO: 70) wascomplementary to the gene sequence from 579^(th) base to 626^(th) baseincluded in the gene fragment complimentary to the scFv gene fragment(SEQ ID NO: 59) except for the seven bases to be substituted. The PCRmethod shown in FIG. 2 allowed the seven bases (AC, TCC, A, C) includedin the expression vector coding for the wild type scFv antibody fragmentto be substituted with the different seven bases (GA, GAT, G, T). Thus,the expression vector containing the gene sequence (SEQ ID NO: 71)encoding the mutant scFv was obtained.

Step (c-3) Acquisition of the Protein Using the Vector

Escherichia coli cells (purchased from Takara bio Co., Ltd, trade name:BL21(DE3)) were transformed with the vector obtained in the step (c-2).Subsequently, the Escherichia coli cells were incubated on an LB plateculture medium containing ampicillin having a concentration of 100 μg/mLunder a temperature of 37 degrees Celsius for 16 hours.

After the incubation, a single colony formed on the LB plate culturemedium was picked up. The single colony was supplied to an LB liquidculture medium containing ampicillin (500 mL) having a concentration of100 μg/mL. Subsequently, the Escherichia coli cells contained in thesingle colony were propagated in such a manner that the absorbance ofthe LB liquid culture medium at a wavelength of 600 nanometers wasadjusted to 0.5.

Furthermore, an aqueous solution of isopropylbeta-D-thiogalactopyranoside (0.5 mL) having a concentration of 1 M wasadded to the LB liquid culture medium. Afterwards, the Escherichia colicells were incubated with shaking under a temperature of 37 degreesCelsius for five hours. In this way, a culture fluid was obtained.

The obtained culture fluid was subjected to a centrifugal separation atan acceleration of gravity of 49000 m/s² under a temperature of 4degrees Celsius for five minutes. The precipitation containing theEscherichia coli cells was again suspended in a phosphate bufferedsaline (50 mL).

The suspension was subjected to an ultrasonic treatment to crush theEscherichia coli cells. The solution containing the crushed Escherichiacoli cells was subjected to a centrifugal separation at an accelerationof gravity of 98000 m/s² under a temperature of 4 degrees Celsius forthirty minutes. In this way, the precipitation was obtained.

The precipitation was washed twice with a phosphate buffered salinecontaining a surface active agent (purchased from Wako Pure ChemicalIndustries Co., Ltd., trade name: Triton X-100) having a concentrationof 4%. The precipitation was further washed with a phosphate bufferedsaline without containing a surface active agent.

An aqueous solution A (10 mL) containing chemical reagents shown inTable 6 was added to the precipitation.

TABLE 6 Chemical reagents Concentration Guanidine hydrochloride  6MSodium chloride 0.1M MES buffer solution 50 mM Ethylene diaminetetraacetic acid 10 mM

The aqueous solution A had a pH of 6.

Subsequently, the aqueous solution A was left under a temperature of 4degrees Celsius for eighteen hours. In this way, the precipitation wasdissolved.

The aqueous solution A was passed through a filter (purchased fromSartorius, trade name: Minisart) having a mesh size of 0.45 μm to removethe residue. In this way, the filtrate was obtained.

Two milliliters of an aqueous solution B was added dropwise to thefiltrate (1 mL). The composition of the aqueous solution B(concentrations of chemical reagents contained in the aqueous solutionB) is shown in Table 7.

TABLE 7 Chemical reagents Concentration Tris-HCl 0.1M Ethylene diaminetetraacetic acid   2 mM Arginine hydrochloride 1.0M Cystamine 3.73 mMCysteamine hydrochloride 6.73 mM

The aqueous solution B had a pH of 8.0. In this way, an aqueous solutionhaving a volume of 3 mL was obtained.

The aqueous solution (3 mL) was added dropwise to an aqueous solutionhaving a volume of one liter which contained the chemical reagents shownin Table 7. Afterwards, the obtained aqueous solution was stirred undera temperature of 4 degrees Celsius for 96 hours. In this way, the mutantscFv antibody fragment (SEQ ID NO: 61) was obtained.

Subsequently, the solution was concentrated using a filtration unit(purchased from Sartorius, trade name: VIVAFLOW50) so that the solutionhad a volume of 10 milliliters. The mutant scFv antibody fragmentcontained in the solution was purified with a column (purchased from GEhealthcare, trade name: HiLoad 26/60 Superdex 75 pg).

The detail of the amino acid sequence (SEQ ID NO: 61) of the mutant scFvantibody fragment is described below.

Light chain variable region (SEQ ID NO: 63):    DVVLTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPLTFGAGTKLEIKR

The amino acid sequence modified at the N-terminal of the light chainvariable region: None

The amino acid sequence modified at the C-terminal of the light chainvariable region: None

Linker (SEQ ID NO: 64): GGGGSGGGGSGGGGSHeavy chain variable region (SEQ ID NO: 65):    QLQLQQSGAELVRSGASVKLSCTASGFNIKDYYMNWVKQRPEQGLEWIGWIDPANGDTAYAPRFQVKADMTADKDSDTAYLQLSSLTSEDTAVYYC NADLPMDQWGQGTSVTVSS

The amino acid sequence modified at the N-terminal of the heavy chainvariable region: None

The amino acid sequence modified at the C-terminal of the heavy chainvariable region:

AAALEHHHHHH (SEQ ID NO: 66)

Step (d) Calculation of Association Rate Constant and Dissociation RateConstant

Using an intermolecular interaction analyzer Biacore T100 (purchasedfrom GE health care company), the association rate constant and thedissociation rate constant of the mutant scFv antibody fragment werecalculated in accordance with the manual attached to the intermolecularinteraction analyzer Biacore T100.

Troponin I (purchased from Funakoshi) derived from human myocardiumhaving approximately 500 RU (Resonance Unit) was fixed on a CM5 chip(purchased from GE health care company). This CM5 chip was set in theBiacore T100. Then, aqueous solutions (concentrations: 100 nM, 50 nM, 25nM, 12.5 nM, and 6.25 nM; volume: 150 microliters) containing the mutantscFv antibody fragment were flowed through the Biacore T100. Theassociation rate constant and the dissociation rate constant measuredwith the intermolecular interaction analyzer Biacore T100 are shown inTable 9.

Comparative Example 1

In Comparative Example 1, the experiment similar to Example 1 wasconducted except that the step (c-2) was not conducted. In this way, thewild type scFv antibody fragment consisting of the amino acid sequencerepresented by SEQ ID NO: 62 was obtained. Similarly to Example 1, theassociation rate constant and the dissociation rate constant of thewild-type scFv antibody fragment were measured. The results are shown inTable 9.

The differences between the wild type scFv antibody fragment (SEQ ID NO:62) and the mutant scFv antibody fragment (SEQ ID NO: 61) are shown inTable 8.

TABLE 8 Wild type scFv Mutant scFv antibody fragment antibody fragment(SEQ ID: 62) (SEQ ID: 61) 197^(th) amino acid in the scFv T D antibodyfragment 203^(rd) amino acid in the scFv S D antibody fragment 205^(th)amino acid in the scFv N D antibody fragment

TABLE 9 Comparative example 1 Example 1 Association rate constant 5.173× 10⁺⁴ 1.24 × 10⁺⁵ Dissociation rate constant 1.361 × 10⁻⁴ 2.14 × 10⁻⁴

As is clear from Table 9, the mutant scFv antibody fragment according toExample 1 has a higher association rate constant than that of thewild-type scFv antibody fragment according to Comparative Example 1.This means that the mutant scFv antibody fragment bound specifically totroponin I derived from human myocardium more quickly than the wild-typescFv antibody fragment.

INDUSTRIAL APPLICABILITY

The recombinant protein and the method according to the presentdisclosure can be used for a sensor for detecting acute myocardialinfarction.

What is claimed is:
 1. A recombinant protein which binds specifically totroponin I derived from human myocardium, the recombinant proteincomprising: a light chain variable region consisting of the amino acidsequence represented by SEQ ID NO: 63; and a heavy chain variable regionconsisting of the amino acid sequence represented by SEQ ID NO:
 65. 2.The recombinant protein according to claim 1, wherein the recombinantprotein is an antibody.
 3. The recombinant protein according to claim 1,wherein the recombinant protein is an antibody fragment.
 4. Therecombinant protein according to claim 3, wherein the antibody fragmentis a Fab antibody fragment.
 5. The recombinant protein according toclaim 3, wherein the antibody fragment is a F(ab′)₂ antibody fragment.6. The recombinant protein according to claim 3, wherein the antibodyfragment is an scFv antibody fragment.
 7. A method for binding arecombinant protein specifically to troponin I derived from humanmyocardium, the method comprising: a step (a) of preparing therecombinant protein, wherein the recombinant protein comprises: a lightchain variable region consisting of the amino acid sequence representedby SEQ ID NO: 63; and a heavy chain variable region consisting of theamino acid sequence represented by SEQ ID NO: 65; and a step (b) ofbringing the recombinant protein into contact with the troponin Iderived from human myocardium to bind the recombinant proteinspecifically to the troponin I derived from human myocardium.
 8. Themethod according to claim 7, wherein the recombinant protein is anantibody.
 9. The method according to claim 7, wherein the recombinantprotein is an antibody fragment.
 10. The method according to claim 9,wherein the antibody fragment is a Fab antibody fragment.
 11. The methodaccording to claim 9, wherein the antibody fragment is a F(ab′)₂antibody fragment.
 12. The method according to claim 9, wherein theantibody fragment is an scFv antibody fragment.